| Esophageal carcinoma is one of the most common malignant tumors in the word. China is a country with a high incidence of Esophageal carcinoma, and Esophageal carcinoma ranks the first in terms of incidence among malignant tumors at present. In recent years, with the application of surgical removal, radiotherapy, chemotherapy and other comprehensive therapeutic measures, there has been a certain degree of progress in the treatment of esophageal carcinoma carcinoma,but the treatment effect is far from being satisfying. As a consequence, it is necessary to be further acquainted with the pathogenesis of esophageal carcinoma to discover more effective therapeutic measures.Human Cripto-1is a cell membrane-anchored protein that has been shown to play an important role in embryonic development and regulating cell differentiation.Cripto-1belongs to the Epidermal Growth Factor (EGF) gene family.The expressed of Cripto-1mainly occurrs in early embryonic development stage.In adult tissues, cells transformed or malignant transformation, which will re-expression. A large number of research results show that Cripto-1is overexpressed in various types of cancers, including nasopharyngeal carcinoma, breast cancer, cutaneous melanoma, oral squamous cell carcinoma. It is involved in several cellular processes linked with neoplasia-like stimulation of cell proliferation, migration, and transformation. Therefore, Cripto-1may be a new tumor-specific markers, targeted blocking the Cripto-1treatment of cancer has become a hot research.However, there is a lack of literature focusing on the effects of Cripto-1in esophageal carcinoma home and abroad. Therefore, this research is intended to inspect and verify the assumption, that is whether there is a correlation between Cripto-1and the genetic development of esophageal carcinoma and what the effect on esophageal carcinoma will be after the blocking of the expression of Cripto-1.In this study, the expression of silence was using RNA interference technology EC9706cells Cripto-1,cells observed Cripto-1protein expression by immunohistochemistry and Western blot, check cell Cripto-1mRNA in situ hybridization and RT-PCTthe expression using the Boden chanm check cell invasion force change for esophageal cancer molecular targeted therapeutic approach to provide the theoretical basis and experimental evidence the expression of Cripto-1protein and mRNA in gastric cancer tissue, atypical hyperplasia tissues and normal gastric mucosa tissues, and analyze its relationship with the invasion and metastasis in gastric carcinoma. Use the RNA interference technology and the CRIPTO-1expression of the silent EC9706cell of gastric cancer, observe the cycle, changes in apoptosis and strength of invasion of cells in gastric carcinoma and the influence on the transplantable tumor in naked mouse with gastric carcinoma.Materials and methods1. Cell culture:esophageal squamous carcinoma EC9706cell line were cultured in Dulbecco’s modified Eagles medium (DMEM) supplemented with10%fetal bovine serum (FBS), incubated in37℃,5%CO2incubation box.2. Select interference target spot, design and synthesize target spot Cripto-1siRNA, transfect Cripto-1siRNA into EC9706cell with lipidosome and act for24,48and72hours respectively. Simultaneously set up blank control (nothing added), negative control (non-specific vector added)(transfection non-specific siRNA)3. Using Western Blot and immunocytochemistry to detect expression of Cripto-1protein of EC9706cells in each groups. 4. Expression of Cripto-1mRNA of EC9706cells in each groups were detected by RT-PCR and in situ hybridization.5.Examine the strength of invasion of cell in each group with Boyden chamber6. Statistical analysis:The SPSS13.0statistical software was used, a=0.05.Results1. The results of Immunohistochemical and Western blotIn contrast with each control group, the expression of Cripto-1protein of each transfection group is invariably lowered and the difference is of statistical significance.(P<0.05) with viscosity and time dependence. The expression of Cripto-1protein is increased along with the increase in the viscosity of transfection and the lengthening of time. The expression differences of Cripto-1protein in each group are of no statistical significance after transfection for48and72hours(P<0.05).2The result of in situ hybridization and RT-PCRIn contrast with each control group, the expression of Cripto-1mRNA of each transfection group is invariably lowered and the difference is of statistical significance.(P<0.05) with viscosity and time dependence. The expression of Cripto-1mRNA is increased along with the increase in the viscosity of transfection and the lengthening of time. The expression differences of Cripto-1mRNA in each group are of no statistical significance after transfection for48and72hours(P>0.05).3The experimental results of Boyden chamber invasion testCompared with the amounts of matrigel gluey cells in blank control group, negative control group and siRNA group.The amount of matrigel gluey cells in blank control group was128.65±6.31, negative control group was123.35±15.12,, and siRNA group was50.24±6.34. The amount of matrigel gluey cells decreased obviously in siRNA group in comparison with blank and negative control group, and the amount differences are of statistical significance (P<0.05)Conclusion:1. Targeted design Cripto-1siRNA can significantly inhibit the expressions of mTOR protein and mRNA in the esophageal squamous cell carcinoma cell line EC9706. It shows that targeted design siRNA Cripto-1can silence Cripto-1gene.2. Cripto-1siRNA can inhibit EC9706cells invasion. It results suggest that it is feasible that Cripto-1siRNA treat esophageal cancer. |
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